Pregnancy in human females can be terminated by unfortunate circumstances, including ectopic pregnancy and spontaneous abortion. Likewise, knowing that a pregnancy is a normal pregnancy with a high likelihood that it will go to term is important, especially to woman who have had previous pregnancies terminated by spontaneous abortion. Detection of the likelihood of either ectopic pregnancy or spontaneous abortion occurring is important to the pregnant woman, in the case of ectopic pregnancy to provide for rapid therapeutic intervention, and in the case of spontaneous abortion to provide an ability to prepare psychologically for the abnormal pregnancy. Failure to diagnose ectopic pregnancy can be harmful and even fatal to the pregnant woman. Yet, it is also highly desirable to distinguish between abnormal pregnancies (i.e., ectopic pregnancies and pregnancies which will spontaneously abort) and normal pregnancies so that desirable normal pregnancies are not needlessly terminated.
Spontaneous abortion is a major health concern to women, affecting about 20% of couples wishing to have children. The diagnostic evaluation of spontaneous abortion is extensive and complex, with many different etiologies, each causing a small proportion of the total cases. The etiologies can be grouped into five categories: anatomic, infectious, hormonal, immunological, and genetic, thereby requiring the collaborative efforts of many medical specialists. Despite such thorough (and expensive) diagnostic workups, it has been estimated that the specific cause for spontaneous remains unknown in a relatively large percentage of affected women (Stephenson, Fertil. Steril. 66:24 (1996).
It is known that if a pregnant woman suffers a spontaneous abortion then any subsequent pregnancies which that woman may have are also more likely to result in spontaneous abortions. The precise reason why certain women suffer such spontaneous abortions is not known but an immunological defect is suspected in many cases of recurrent spontaneous abortion for which no underlying genetic, hormonal, microbiological or anatomical abnormality has been detected. This defect is believed to be related to a failure of maternal immune recognition of the implanting blastocyst, which results in the lack of production of protective factors and subsequent rejection of the developing foetal trophoblastic tissue at maternal-foetal interfaces. Pregnancy outcome for these women is especially precarious and attempts to provide some insight into the outcome of pregnancy would be desirable benefit for these women.
The incidence of ectopic pregnancy in the United States has risen 6-fold in the last three decades. According to the 2002 American College of Obstetrics and Gynecology Survey, ectopic pregnancies now account for about 2.1% of reported pregnancies. Despite the available knowledge with regard to progesterone and hCG levels in pregnant females, rapid screening tests at the point of care for confirming the existence or likelihood of an ectopic pregnancy or spontaneous abortion are not generally available. Thus, it would be desirable to provide rapid screening assays which could be utilized in blood, plasma, serum or urine samples from the pregnant female to confirm the continuing normal nature of the pregnancy or to assess the likelihood of an abnormal pregnancy, i.e., an ectopic pregnancy or spontaneous abortion. Diagnostic tests are now needed to identify a normal or abnormal pregnancy at an early stage rapidly and to a high degree of certainty, especially when a pregnant woman has experienced recurring spontaneous abortion or the patient exhibits symptoms of ectopic pregnancy or other complications of pregnancy before it is clinically evident. Such tests should be rapid, highly accurate and preferably taking no more than an hour or less to run, and should be convenient so that the tests could be performed at the point of care.
This would be useful in all pregnancies and especially those in pregnant women who have had at least one spontaneous abortion. This would also be useful at point-of-care facilities or in home pregnancy tests. Presented with possible complications of pregnancy, the ER or point-of-care physician would among other tests perform a POC hCG pregnancy test as well as a test to determine pregnancy outcome, i.e., to determine the likelihood that any pregnancy is a normal pregnancy or abnormal pregnancy (e.g., ectopic or spontaneous abortion). If the pregnancy test is positive, it would then be a case of trying to differentiate a normal intrauterine pregnancy from an extrauterine pregnancy (ectopic) or a pregnancy likely to result in a spontaneous abortion. There are approximately 100,000 proven ectopic pregnancies (in 5 million pregnancies) in the USA each year and up to a million or more pregnancies which spontaneously abort. The incidence (actual numbers) of these pregnancies is much higher outside the United States.
Currently, there is no single fast, accurate assay for assessing a normal pregnancy outcome at an early stage in a pregnant woman. There are complicated tests for determining the existence of ectopic pregnancy. In the case of ectopic pregnancy, the approach for determining its existence requires that a woman who presents with ectopic pregnancy consistent symptoms undergoes a two-step analysis which uses transvaginal ultrasound coupled with an hCG doubling test to evidence the existence of ectopic pregnancy. In the case of transvaginal ultrasound, this is used to see if there is evidence of a fetus by looking for a fetal sac. A transabdominal ultrasound may be used to see if there is evidence of ectopic pregnancy. The problem with using this approach is in early pregnancies the fetus or fetal sac, at 5-8 weeks or pregnancy (usually, about 1-4 weeks after missing of menses), the time when symptomology of ectopic pregnancy first becomes evident, is miniscule and may often be missed using ultrasound. In addition to the use of transvaginal ultrasound, the analysis also relies on the use of the hCG doubling test to see if serum hCG results double or more than double in 2 days. If hCG does not double within the two day period, that is an indicator of ectopic pregnancy. In contrast, in the case of spontaneous abortion, there is no current approach for determining its likelihood.
As described above, the conventional approach(es) for differentiating a normal pregnancy from an abnormal pregnancy, whether that abnormal pregnancy is an extraurterine ectopic pregnancy or a spontaneous abortion are either deficient, or, in the case of spontaneous abortion, virtually non-existence. As such, mistakes are often made and normal and abnormal pregnancies are wrongly diagnosed. With respect to woman who have had at least one spontaneous abortion, determining that a pregnancy at an early stage is a normal pregnancy is an important issue. Another important issue is to identify the likelihood of abnormal pregnancy, rapidly and with a high degree of accuracy, so that monitoring of the pregnancy or treatment may begin expeditiously.
Human chorionic gonadotropin (hCG), a glycoprotein hormone, exists in pregnancy urine and serum as early as the week following implantation (1-6). Maternal concentrations of hCG are widely used as a means of distinguishing normal and abnormal pregnancies, with the concentration of maternal hCG reportedly doubling approximately every 2 days in the first 2 months of a favorable outcome gestation (7-9). Measuring the hCG doubling rate over 2 days has become a standard part of prenatal care. Falling serum hCG results, or concentrations that do not double within two days, are used to indicate a failing pregnancy, either a spontaneous abortion in the first trimester of pregnancy or ectopic pregnancy. hCG levels that at least double over 2 days indicate a favorable outcome pregnancy, that likely will go to term (9). Early identification of a failing pregnancy can help ensure proper care and management; this is particularly crucial in the case of an ectopic pregnancy.
The hCG doubling (in two days) test is, however, as explained above, awkward. Firstly, the need to make two office/clinic visits, to order the test and discuss the result with the physician. Secondly, by the need to make 2 laboratory visits or have 2 blood draws over exacting times. Furthermore, as reported, this test is limited in sensitivity (depending on the report, 62-78%), and by a high rate of false-positive rate (26-40%) (10-11). It is the experience of the University of New Mexico Reproductive Endocrinology Clinic, for instance, that this test detects 73% of failures at a 40% false-positive rate (Byrn et al., unpublished data). Considering the reported risks in the USA of spontaneous abortion and ectopic pregnancy (12-15), this test has a predictive value positive (for pregnancy failures) of 35%. Much more accurate tests are needed to assess risk of pregnancy failure and favorable outcomes, preferably with greater ease-of-use.
Hyperglycosylated hCG (“H-hCG”) is an hCG molecule with additional sugar residues on its oligosaccharide side chains (16-18). Specific antibodies have been raised against H-hCG and assays have been established to detect only H-hCG (19). An FDA-approved automated chemiluminescence immunoassay for H-hCG is now available commercially, and used throughout the USA (20).
H-hCG has been shown by multiple authors to be the predominant form of hCG present in serum and urine samples in early pregnancy, during the time when implantation is occurring and the month that follows (21-26). It has been previously shown that significantly lower proportions of H-hCG (vs. hCG) are found in spontaneously aborting and ectopic pregnancies (25, 26). Considering the lower concentrations of hCG in failing pregnancies (7, 8, 10, 11), and the lower proportions of H-hCG (16-18), measurement of H-hCG concentrations may provide an amplified means of detecting failing pregnancies. However, no parameters have be described for measuring H-hCG to differentiate favorable outcome and pregnancy failure.
The present invention is directed to improved means for evaluating pregnancy outcome. We investigated H-hCG measurement protocols for serum and urine samples. Urine for potential over-the-counter and point-of-care applications and serum for point-of-care and professional laboratory applications. Urine samples are preferably those obtained initially upon waking of the patient/subject. Sensitivity. specificity, predictive values and diagnostic accuracy are assessed of single sample H-hCG measurements, and compared with analogous hCG results.